Improved Multiprotein Microcontact Printing on Plasma Immersion Ion

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Improved multi-protein micro-contact printing on plasma immersion ion implanted polystyrene Elena Kosobrodova, Wan Jun Gan, Alexey Kondyurin, Peter Thorn, and Marcela M.M. Bilek ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15545 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

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ACS Applied Materials & Interfaces

Improved multi-protein micro-contact printing on plasma immersion ion implanted polystyrene

E. Kosobrodova1*, W. J. Gan2*, A. Kondyurin1, P. Thorn2#, M. M. M. Bilek1,2,3,4#

1

2

The School of Physics, University of Sydney, Sydney, NSW, Australia, 2006 Department of Physiology, Sydney Medical School, Charles Perkins Centre, University of

Sydney, Sydney, NSW, Australia, 2006 3

The School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney,

NSW, Australia, 2006 4

The Australian Institute of Nanoscale Science and Technology, University of Sydney, Sydney,

NSW, Australia, 2006 #

Equal senior authors

*

Corresponding authors

KEYWORDS: multi-protein micropattern, plasma immersion ion implantation, pancreatic eta cells, phospho-paxillin

ACS Paragon Plus Environment

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ABSTRACT Multi-protein micropatterning allows the creation of complex, controlled microenvironments for single cells that can be used for the study of the localized effects of various proteins and signals on cell survival, development and functions. To enable analysis of cell interactions with microprinted proteins, the multi-protein micropattern must have low cross-contamination and high long-term stability in a cell culture medium. To achieve this, we employed an optimized plasma ion immersion implantation (PIII) treatment to provide polystyrene (PS) with the ability to covalently immobilize proteins on contact whilst retaining sufficient transparency and suitable surface properties for contact printing and retention of protein activity. The quality and long-term stability of the micropatterns on untreated and PIII treated PS were compared with those on glass using confocal microscopy. The protein micropattern on the PIII treated PS was more uniform and had a significantly higher contrast that was not affected by long term incubation in cell culture media because the proteins were covalently bonded to PIII treated PS. The immunostaining of mouse pancreatic β cells interacting with E-cadherin and fibronectin striped surfaces showed phosphorylated paxillin concentrated on cell edges over the fibronectin stripes. This indicates that multi-protein micropatterns printed on PIII treated PS can be used for high resolution studies of local influence on cell morphology and protein production.

1.

Introduction


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Cell survival and functions in vivo are regulated by complex cell-cell and cell-extracellular matrix (ECM) interactions.1 Multi-protein micropatterning allows replication of a single cell microenvironment and the study of the effects of various biomolecules on cell attachment,2, 3 spreading,2,

4

apoptosis

5

and other functions.6,

7

For tissue engineering, multi-protein

micropatterning provides a method to spatially control cell growth and secretory output.8,

9

However, most of the existing micro-contact printing (µCP) platforms rely on a combination of adhesive and non-adhesive regions

10

and are limited to a single protein layer per pattern,5, 10 so

they cannot adequately mimic complex multiprotein cell microenvironments. The proteins printed on unmodified glass and polymers are physically adsorbed. During washing steps, the micropatterns of such physisorbed proteins tend to fuse, smear or be washed away completely.11 The long-term stability of these micropatterns in a protein-rich solution is very low

12

due to

competitive protein exchange known as the Vroman effect.13 This issue is even more problematic in the case of multi-protein micropatterns where instability under prolonged cell culturing

14

is

accompanied by a high level of cross-contamination.15 Although a number of studies have been reported on multi-protein micropatterning,2, 11, 16-19 long-term stability of these micropatterns in cell culture was not tested. The proteins used for multi-protein micropatterning in these studies were physisorbed or covalently attached to physisorbed bovine serum albumin (BSA). The longterm stability of a protein micropattern can be significantly improved by covalent immobilization of proteins directly to the substrate. For example, silanizing the glass surface with (3aminopropyl)triethoxysilane provides glass with an ability to irreversibly bind protein through amine groups.20 However, the most of the chemicals used for silanization are cytotoxic and their residuals have a negative effect on cell survival.21, 22


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An alternative method of surface modification not requiring any chemical linkers for covalent immobilization of biomolecules, recently reviewed in (PIII).24,

25

23

is plasma immersion ion implantation

PIII treated polymer surfaces have a significantly improved wettability,26 protein

binding capacity,27 and cytocompatibility

28-30

than unmodified polymer surfaces. In addition,

proteins form a denser layer 25 with a higher bioactivity 27 and a better-preserved conformation 31 on PIII treated surfaces compared to the proteins attached to untreated polymers. The use of plasma deposited 32, 33 and plasma ion implanted 34 polymers as a platform for µCP allows welldefined micropattern to be obtained. However, the long-term stability of these patterns in a protein rich solution has not been tested to date either.

Compared to UV- and γ-irradiation and conventional plasma treatment, PIII treatment creates a significantly higher concentration of long-lived radicals in the modified layer.35 These radicals are believed to be responsible for covalent protein attachment

24

to the ion modified surface.

Therefore, the PIII treated surface has a higher protein binding capacity and longer activity for covalent protein immobilization than a surface treated by plasma alone.36

In this paper, we analyze uniformity, contrast and long-term stability of multi-protein micropatterns printed using PDMS stamps on untreated and PIII treated PS as well as on glass coverslips. In order to verify that our approach yields micropatterns that are stable in long term cell culture and can locally affect cellular responses, we have used insulin-secreting pancreatic β cells as a model system. In native islets of Langerhans pancreatic β cells make complex interactions with other endocrine cells 37 and with the ECM of the capillary bed.38 Understanding


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the nature of these responses, is informing the development of cell-based therapies for type 1 diabetes. For example, by enhancing islet function in islet transplantation 39 and leading to better supportive environments for stem cell derived β cells.40

Recent studies have shown that β cells, within intact islets of Langerhans, are structurally 41 and functionally polarized with respect to the vasculature.42 This establishes multiple membrane domains in each β cell.41 The mechanism of this orientation is unknown but it is likely to be driven by extracellular cues. Culture of isolated β cells on single substrates shows the adherens junction protein, E-cadherin,37 and ECM proteins, like fibronectin

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do affect cell function.

Here, we use micropatterns of fibronectin and E-cadherin stripes, with a width of few micrometers, to closely replicate the multiple cues that are presented simultaneously to single β cells within the islet.

We show that the protein pattern on PIII treated PS surfaces triggers spatially discrete responses in the β cells and will enable us to determine the mechanisms behind the establishment of these local domains that are likely to be very significant for β cell function and the control of insulin secretion.

2.

Methods and Materials


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2.1. Spin-coating

2 % w/v solution of PS (BASF, Australia) in toluene (Sigma-Aldrich, Australia) was spin-coated onto (100) borosilicate glass coverslips (Menzel-Gläser, Germany) or silicon wafers (Topsil, USA) using an SCS G3P-8 Spin-coater with a speed of 2000 rpm. The silicon wafers were Pdoped (104 – 2·104 Ω·cm) and had one side polished. Their thickness was 0.610–0.640 mm. Before the spin-coating, the silicon wafers were washed for 20 s in mQ-water with ultrasound and air dried. To improve adhesion of the PS film to the glass surface, glass coverslips were plasma treated for 10 min prior to the spin-coating.

2.2. Plasma ion implantation

Surface modification of PS coated glass coverslips was performed using inductively coupled radio-frequency (RF) nitrogen plasma, powered at 13.56 MHz. RF plasma was used as a source for PIII. The base pressure of the vacuum system was 10-5 Torr (10-3 Pa) and the pressure of nitrogen gas used for PIII was 2·10-3 Torr (0.267 Pa). The forward power was 100 W with reverse power of 12 W when matched.


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The samples were placed on a stainless steel holder with a mesh made of the same material. The mesh was electrically connected to the holder and held in front of the sample parallel to its surface. The distance between the sample and the mesh was 5 cm. Plasma ions were accelerated by the application of high voltage (20 kV) negative bias pulses of 20 µs duration at a frequency of 50 Hz to the substrate holder and its mesh. The biased holder and mesh drew a current of 1.3 mA. The spin-coated PS films were PIII treated for 80 s and 800 s corresponding to ion implantation fluences of 1015 ions/cm2 and 1016 ions/cm2, respectively. The samples were stored for a week in air at room temperature before protein microcontact printing.

2.3. Ellipsometry

The thicknesses and optical constants of untreated and PIII treated PS spin-coated on silicon were measured using a Woollam M2000V spectroscopic ellipsometer. Ellipsometric data were collected for three angles of incidence: 65°, 70° and 75°. A three-layer model was used to fit the data collected from the samples before protein microcontact printing. The layers were silicon, native silicon oxide and a Cauchy layer representing the PS film. For the samples with a protein coating, a four-layer model was used and the second Cauchy layer corresponded to the protein layer. The thickness and optical constants associated with the best fits were determined for each sample.

2.4. Wettability


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The contact angles of de-ionized water and diiodomethane on untreated and RF plasma treated glass coverslips as well as untreated and PIII treated PS were measured 1 h and 7 days after the treatment using a Kruss contact angle analyzer DS10 employing the sessile drop method. Average values were calculated using 3-5 measurements for each sample. The error of the contact angle measurements was 0.3° - 1.5°. The water contact angles of untreated and oxygen plasma treated poly(dimethylsiloxane) (PDMS) stamps (Bandwidth Foundry, Australia) were measured 1 h after the plasma treatment. Surface free energy and its dispersive and polar components were calculated using the Owens-Wendt-Rabel-Kaelble model.

2.5. X-ray photoelectron spectroscopy (XPS)

Chemical composition of untreated/RF plasma treated glass coverslips and untreated/PIII treated PS films spin-coated on the RF plasma treated glass coverslips was analyzed using an X-ray photoelectron spectrometer equipped with Al Kα X-ray source (1486.6 eV, Specs, Germany) with monochromator operating at 200 W, a hemispherical analyser (Phobios 100, Specs, Germany) and a line delay detector with 9 channels. Survey spectra were acquired for binding energies in the range from 0 to 1200 eV using 30 eV pass energy. C 1s, O 1s and N 1s region spectra were acquired at a pass energy of 23 eV with 10 scans to obtain high spectral resolution at low noise level. The C1s peaks of 80 s and 800 s PIII treated PS were fitted with a sum of five Lorentz functions using the Marquardt-Levenberg fitting procedure of Casa XPS. The peaks were quantified using relative sensitivity factors supplied by the spectrometer manufacturer. A


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linear background was subtracted and the spectra were charge corrected by setting the C 1s CC/H component to 285.0 eV.

2.6. Fourier transform infrared - attenuated total reflectance (FTIR-ATR) spectroscopy

FTIR-ATR spectra of untreated and PIII treated PS films (Goodfellow, Cambridge, UK) incubated in bovine serum albumin (BSA) (Sigma-Aldrich, Australia) and phosphate buffered saline (PBS) (Sigma-Aldrich, Australia) were measured using a Digilab FTS7000 FTIR spectrometer fitted with a multibouncing ATR accessory (Harrick, USA) with a trapezium germanium crystal at an incidence angle of 45°. To obtain sufficient signal/noise ratio and resolution of spectral bands, 1000 scans were taken at a resolution of 4 cm-1.

Untreated and PIII treated PS were incubated for 1.5 h at 23 °C in 5% w/v BSA. Incubation of PIII treated PS was performed on the next day and 11 days after the PIII treatment. After the incubation in BSA, the samples were rinsed in PBS (pH 7.4), placed into fresh PBS for 1.5 h and rinsed four times in mQ-water. Two other samples of untreated and PIII treated PS were incubated for 3 h in PBS, then rinsed four times in mQ-water and used as controls.

To calculate the amount of immobilized protein, FTIR-ATR spectra of the samples incubated in PBS were subtracted from the spectra of the corresponding protein coated samples. The


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difference spectra were normalized on the intensity of the 1601 cm-1 PS vibration line of the corresponding spectra of protein-coated samples. The intensities of Amide A and Amide II peaks were normalized by 0.14 and 0.47, respectively

44

. Then, the average intensity of Amide I,

normalized Amide II and Amide A peaks were calculated together with the standard deviation.

2.7. Covalent protein attachment test

To test covalent attachment of BSA, untreated and PIII treated PS incubated in BSA and PBS were washed for 1 h in 5% sodium dodecyl sulfate (SDS) (Sigma-Aldrich, Australia) at 70 °C. After the SDS washing, the samples were rinsed in mQ-water, washed for 30 min in fresh mQwater and rinsed in mQ-water again.

FTIR-ATR spectroscopy cannot be used for analysis of protein attachment to the surface of a glass coverslip due to the very intensive absorbance of glass in the low wavenumber region. The effect of SDS washing on the amount of BSA attached to a glass surface was studied using XPS. The nitrogen concentration was chosen as an indicator of protein attachment. Four glass coverslips were incubated in 5% w/v BSA or PBS as described in the Section 2.6. Two glass coverslips incubated in BSA or PBS were SDS washed for 1 h at room temperature, rinsed in mQ-water, washed for 30 min in fresh mQ-water and rinsed in mQ-water again. All samples were dried overnight at room temperature prior to FTIR-ATR and XPS analysis.


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2.8. Protein microcontact printing

A PDMS stamp with 2 µm parallel ribs separated by 5 µm spaces (Bandwidth Foundry, Australia) was cut to 0.5 cm x 0.5 cm and cleaned by sonication in 80% ethanol (Sigma-Aldrich, Australia) for 3 minutes. After rinsing with mQ-water, the stamp was air-dried and oxygen plasma treated at 200 W power and 70 mTorr pressure for 3 minutes in a PC2000 plasma cleaner (South Bay Technology, San Clemente, CA, USA). Then, the stamp was inked with 5 µl of a first layer protein (50 µg/ml fibronectin or 50 µg/ml E-cadherin) for 30 minutes. Extracellular matrix protein, fibronectin (F5147, Sigma-Aldrich, Australia) was conjugated with FITC using the fast FITC conjugation kit (ab188285, Abcam, Melbourne, VIC, Australia) and used as the first layer protein in the micropattern quality and stability studies. Recombinant mouse Ecadherin Fc chimera protein (748-EC, R&D Systems USA) was used as the first layer protein in the study of cell attachment and phospho-paxillin production. After rinsing in PBS and blotting of excess solution, the stamp was inverted and pressed to the surface of a glass coverslip or PS for 10 minutes. A 30 g weight was placed on the top of the stamp to ensure a good contact with the surface. After removing the stamp, the micropatterned surface was incubated with a second layer of protein, Alexa Fluor 555-conjugated bovine serum albumin (10 µg/ml, BSA-555, A34786, Thermo Fisher Scientific, Australia) or 10 µg/ml fibronectin for 15 minutes. BSA-555 was used as backfilling protein in the micropattern quality and stability studies, while fibronectin was used as the backfilling protein in the study of cell attachment and phospho-paxillin production. Lastly, the micro-patterned surface was rinsed in PBS and stored in PBS at 4 °C until cell seeding.


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2.9. Islet isolation and dispersion

C57BL/6 mice were humanely sacrificed according to the local University of Sydney ethics procedures approved by the Animal Ethics Committee. Islets were isolated as previously described.45 Briefly, the pancreas was inflated and digested with an enzyme mixture of liberase TL (Roche, Australia) and collagenase type-IV (Life Technologies, Australia), followed by separation of islets from tissue debris using histopaque 1077 (Sigma-Aldrich, Australia). Islets were placed into culture media RPMI-1640 supplemented with 10% fetal bovine serum (Life Technologies, Australia) and 1% penicillin-streptomycin (Life Technologies, Australia) and left to recover in the cell culture incubator (37°C, 95/5% air/CO2) for 1 hour. Then, islets were rinsed in PBS and dispersed in 1 ml heated trypLE express enzyme (Life Technologies, Australia) for 3 minutes with gentle shaking. After centrifuging and removing the enzyme solution, dispersed cells were resuspended in culture media and seeded onto the micro-patterned surface. The cells were incubated overnight (37°C, 95/5% air/CO2).

2.10. Immunostaining

Cells were fixed in ice-cold 4% w/v paraformaldehyde (Sigma-Aldrich, Australia) in PBS for 15 minutes, blocked for 1 hour with a PBS based buffer consisting of 3% BSA, 3% donkey serum (Sigma-Aldrich, Australia) and 0.1% triton X-100 (Scharlau Chemicals, Australia) diluted in


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PBS and incubated with primary antibody (phospho-paxillin Tyr118, #2541, 1:200 dilution, Cell Signaling Technology, USA) overnight at 4°C. After washing with PBS, cells were incubated with secondary antibody (donkey anti-rabbit Alexa Fluor 546, A10040, 1:200 dilution, Life Technologies, Australia) for 30 minutes at room temperature. Coverslips with fixed, stained and washed in PBS cells were mounted in CitiFluor AF2 antifadent (ProSciTech, Kirwan, QLD, Australia) and the edge was sealed with nail polish.

2.11. Relative transmittance reduction of untreated and PIII treated PS

Reduction of visible light transmittance of untreated and PIII treated PS films spin-coated on glass coverslips relative to transmittance of a glass coverslip was measured by taking brightfield images using a Zeiss Axio VertA1 inverted light microscope and analyzing their pixel intensity using ImageJ. Light intensity of untreated and PIII treated PS films was normalized on light intensity of a glass coverslip.

2.12. Autofluorescence measurements and imaging

Autofluorescence measurements and fluorescent images were taken using a Leica TCS SP8 inverted confocal microscope equipped with a white light laser and acousto-optic beam splitter system. Leica HCX PL APO 40x/0.85 CORR CS or HC PL APO 63x/1.40 Oil CS2 objectives were used.


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The autofluorescence of glass coverslips and untreated/PIII treated PS films spin-coated on glass coverslips was measured using lambda scans in the 440nm – 765nm range at the laser excitation wavelengths of 488nm, 553nm, and 631nm. Excitation/emission settings of ex488nm/em500nm676nm and ex553nm/em564nm-703nm were used for the micro-pattern imaging. Minimum intensity across the field of view was taken as the background and subtracted from the images. The images were quantified using fluorescence linescan with the value normalized to the maximum intensity. The non-specific binding among two different proteins was determined using the intensity ratio between adjacent stripes.

For the cell imaging, the settings of ex499nm/em509nm-601nm and ex557nm/em567nm-687nm were used. The area of phospho-paxillin on each stripe was quantified using a threshold on the fluorescence intensity to produce a binary image. The area occupied by phospho-paxillin within each cell was measured (Image J) and normalized against the total area of the cell footprint on an individual stripe.

2.13. Statistics

The error bars in Figs. 2 and 4 represent standard deviations. The error bars in Fig. 3 show the uncertainty of XPS measurements. All other data were presented as mean ± standard error mean


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(SEM). Statistical analysis was performed using one-way ANOVA, student paired or unpaired ttest. Statistical significance was indicated as * p

Improved Multiprotein Microcontact Printing on Plasma Immersion Ion

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